Guanidine derivatives as combined thromboxane A2 receptor antagonists and synthase inhibitors.

A new series of omega-disubstituted alkenoic acid derivatives derived from samixogrel 5 were designed and synthesized as combined thromboxane A2 receptor antagonists/thromboxane A2 synthase inhibitors with improved solubility and reduced protein binding compared to 5. Hexenoic acid derivatives with a 3-pyridyl group and 3-(2-cyano-3-alkyl-guanidino)phenyl substituent were found to be optimal with regard to this dual mode of action. The most potent compound, E-6-(3-(2-cyano-3-tert-butyl-guanidino)phenyl)-6-(3-pyridyl)hex-5-eno ic acid, "terbogrel" 32 inhibits the thromboxane A2 synthase in human gel-filtered platelets with an IC50 value of 4.0 +/- 0.5 nM (n = 4). Radioligand binding studies with 3H-SQ 29,548 revealed that 32 blocks the thromboxane A2/endoperoxide receptor on washed human platelets with an IC50 of 11 +/- 6 nM (n = 2) and with an IC50 of 38 +/- 1 nM (n = 15) in platelet-rich plasma. Terbogrel inhibits the collagen-induced platelet aggregation in human platelet-rich plasma and whole blood with an IC50 of 310 +/- 18 nM (n = 8) and 52 +/- 20 nM (n = 6), respectively. This was shown to translate into a potent antithrombotic effect in vivo as demonstrated in studies using a model of arterial thrombosis in rabbits (ED50 = 0.19 +/- 0.07 mg/kg; n = 20). Thus, terbogrel is the first compound with a guanidino moiety demonstrating both a potent TXA2 synthase inhibition and a potent TXA2 receptor antagonism and has been selected for further clinical development.

PK/PD simulations as a tool for rational design of clinical dosage regimens: an example with Fradafiban.

In clinical studies, pharmacodynamic effects should be achieved, e.g. maintenance of certain effects with reversibly acting drugs. Available data base for early phase II studies is frequently kinetics in healthy volunteers from phase I studies and pharmacodynamic effects from in vivo or ex vivo data. Using the fibrinogen receptor antagonist Fradafiban as an example, a procedure to achieve rational dosage regimens is described. Applied methods were: curve fitting of plasma concentrations obtained in phase I studies, correlation of fibrinogen receptor occupancy (FRO) to plasma concentrations using a sigmoid Emax model with Hill coefficient for PK/PD correlation, simulation of time course of FRO for various dosage regimens, using estimates for variability from PK and PD data in order to estimate not only mean values but also expected range of FRO. In a phase II study with Fradafiban administered intravenously, therapeutically active plasma levels had to be achieved rapidly and maintained over 24 hours employing a simple infusion regimen in 20 patients and 3 dose groups. For the target dose, a FRO of 80% should be achieved in most patients. Using the tools mentioned above, a rapid initial infusion rate of 10 mg over 30 minutes, followed by a maintenance infusion of 30 mg for the remaining 23.5 hours for the target dose resulted in a median predicted FRO of 82%. For the lower dose, a 5/15 mg infusion over 0.5/23.5 hours, achieving a predicted FRO of 68% was used and the upper dose (15/45 mg) resulted in 87% FRO. Median experimental results were 84% FRO for the target dose and 73% and 88%, respectively, for the lower and upper dose. As these results fit reasonably well to the predictions, it can be concluded that kinetics in the mostly elderly patients is similar to kinetics in healthy young volunteers. Furthermore, FRO in patients is nearly identical to that in spiked human plasma. Taken together, it could be proven that PK/PD methods are a useful tool for design of clinical studies of Fradafiban.

Profound and sustained inhibition of platelet aggregation by Fradafiban, a nonpeptide platelet glycoprotein IIb/IIIa antagonist, and its orally active prodrug, Lefradafiban, in men.

BACKGROUND: Clinical trials have demonstrated that platelet glycoprotein (GP) IIb/IIIa antagonists effectively prevent acute thrombotic events. Orally active GP IIb/IIIa antagonists are essential to evaluate the clinical benefit of long-term treatment. We therefore investigated platelet inhibition by the GP IIb/IIIa antagonist Fradafiban (BIBU 52; Fradafiban is the recommended INN of BIBU 52) and its orally administered prodrug, Lefradafiban (BIBU 104; Lefradafiban is the recommended INN of BIBU 104) in healthy subjects. METHODS AND RESULTS: The activity and plasma levels of Fradafiban and Lefradafiban were evaluated in double-blind, placebo-controlled studies in 130 healthy male subjects. One to 15 mg Fradafiban continuously infused over 30 minutes reversibly inhibited platelet aggregation in platelet-rich plasma ex vivo in response to 20 micromol/L ADP (5 mg, 100% inhibition at 27 minutes after administration) and to both 1.0 (5 mg, 100%) and 10 microg/mL (15 mg, 97+/-3%) collagen. Single oral doses of Lefradafiban inhibited ADP-induced aggregation by 59+/-14% (50 mg [mean+/-SD]; n=8), 90+/-12% (100 mg), and 99+/-2% (150 mg) 8 hours after administration. Correlations between activity and Fradafiban plasma levels were identical after Fradafiban and Lefradafiban treatment. After day 1, oral TID Lefradafiban treatment for 7 days inhibited aggregation by > or = 31+/-9.6% (25 mg TID; n=8), 53+/-12% (50 mg; n=7), and 88+/-6.6% (75 mg; n=8) just before the next dose. A similar correlation between the activity and Fradafiban plasma levels was observed at days 1, 2, and 7. CONCLUSIONS: Oral administration of Lefradafiban maintains the potent platelet GP IIb/IIIa antagonism of Fradafiban during treatment of healthy subjects for 1 week without signs of loss of the antiplatelet activity.

Antagonism of the GPIIb/IIIa receptor with the nonpeptidic molecule BIBU52: inhibition of platelet aggregation in vitro and antithrombotic efficacy in vivo.

The glycoprotein (GP) IIb/IIIa (the alphallb beta3 integrin) found on platelets binds fibrinogen or von Willebrand factor when the platelet is activated, thereby mediating the aggregation of platelets. Blockade of the GPIIb/IIIa should prevent platelet aggregation independent of the substance or substances responsible for activating the platelets. This comprehensive inhibition of platelet aggregation is thought to be an effective therapeutic approach to various clinical thromboembolic syndromes. This study investigated the platelet inhibition provided by blocking GPIIb/IIIa by using a new nonpeptidic molecule, BIBU52, in both in vitro and in vivo models. BIBU52 competes with [125I]fibrinogen for binding sites on human platelets in a Ca2+ and pH-dependent manner with a 50% inhibitory concentration (IC50) of 35 +/- 12 nM. BIBU52 inhibited the aggregation of human platelets in platelet-rich plasma induced by collagen (1-2 microg/ml), adenosine diphosphate (ADP; 2.5 microM), and a thrombin receptor-activating peptide (TRAP; SFLLRNPNDKYEPF-NH2; 25 microM) with IC50 values of 82, 83, and 200 nM, respectively. The inhibition of platelet aggregation by BIBU52 was found to be highly species dependent. BIBU52 inhibited aggregation in plasma from rhesus and marmoset monkeys with an IC50 of 150 nM but was totally ineffective in rat plasma. The selectivity of BIBU52 for inhibiting GPIIb/IIIa in comparison with other adhesion molecules was investigated in a human endothelial cell adhesion assay. The adhesion of human endothelial cells to matrices of vitronectin, fibronectin, collagen I, or laminin was not affected by concentrations as high as 100 microM BIBU52; thus BIBU52 demonstrates a high selectivity for the human GPIIb/IIIa. The antithrombotic effect of BIBU52 in vivo was investigated in three animal models of recurrent arterial thrombus formation. In the guinea pig aorta, BIBU52 inhibited thrombus formation dose dependently, with lack of thrombus formation for 1 h after a bolus dose of 1.0 mg/kg i.v.. Both acetylsalicylic acid and dazoxiben were less effective in this model. In pigs with recurrent thrombus formation in the carotid artery, 1.0 mg/kg i.v. also inhibited thrombus formation. Heparin was not effective in the pig, and acetylsalicylic acid was only partially effective. In the pig, the dose of 1.0 mg/kg i.v. BIBU52 also was associated with a 70% inhibition of collagen-induced platelet aggregation ex vivo but with only a transient prolongation of sublingual bleeding time to a maximum of 2.5-fold and without other hemodynamic effects. In the marmoset monkey, a dose of 10 microg/kg i.v. could abolish recurrent arterial thrombosis. Hemodynamic effects of BIBU52 in anesthetized pigs were not detected in doses < or = 10 mg/kg. These data demonstrate that BIBU52 is a potent and selective antagonist of the human GPIIb/IIIa receptor and capable of substantial inhibition of platelet aggregation in vitro and ex vivo as well as inhibition of arterial thrombus formation in vivo in animal models of thrombosis.

Inhibition of platelet aggregation as a surrogate marker.

Using acetylsalicylic acid-dipyridamole, a combined thromboxane receptor antagonist-thromboxane synthase inhibitor, and a fibrinogen receptor antagonist as examples, this article discusses the predictive value of several methods that may be employed in the evaluation of antiaggregatory effects and their suitability as surrogate markers for the planning of patient studies. Platelet aggregation in various ex vivo tests and the effects of drugs on these tests were investigated using platelet aggregation in platelet-rich plasma and in whole blood, and in thrombus formation on a thrombogenic surface. Drug-induced inhibition of platelet aggregation is based on the modulation of metabolic processes or interactions at the membrane-receptor level. These effects must be assessed by methods adapted specifically to each mode of action, for example, mediator synthesis inhibition (cyclooxygenase), thromboxane receptor antagonism-thromboxane synthase inhibition, or fibrinogen receptor blockade. Thus a combination of both general and specific methods adapted to the respective mode of action of the test substance can serve as surrogate markers of drug efficacy for the efficient planning of clinical trials.

Pharmacokinetic-pharmacodynamic and metabolite modeling with TopFit.

Two examples illustrating the ease of use and powerful data fitting and simulation techniques provided by the validated program TopFit 2.0 for the PC are presented. In the first, kinetics of parent compound and metabolite for (+) and (-) enantiomers of a racemic compound X were determined during a Phase III clinical study. Four data sets were fitted simultaneously for each patient. The model could be defined by the user without programming differential equations. The fit results indicated enantiomer specific kinetics for the metabolite but not for parent compound. In the second example, a model with nonlinear elimination and an Emax-effect function was used to simultaneously fit data from six doses of compound Y in a Phase I study. The fitted parameters predicted the feasibility of a twice-daily dose regimen despite a very short plasma half-life of the compound. In conclusion, TopFit provides rapid and cost-effective support in analysis and design of clinical trials.

6,6-Disubstituted Hex-5-enoic acid derivatives as combined thromboxane A2 receptor antagonists and synthetase inhibitors.

A series of omega-disubstituted alkenoic acid derivatives were designed and synthesized as antithrombotic inhibitors of thromboxane A2 synthetase and thromboxane A2 receptor antagonists. Hexenoic acid derivatives with a 3-pyridyl group and a 4-(2-benzenesulfonamidoethyl)phenyl substituent were found to be optimal with regard to the dual mode of action. The most potent compound, (E)-6-(4-(2-(((4-chlorophenyl)sulfonyl)amino)ethyl)phenyl)-6-(3-pyridyl) hex-5-enoic acid (36), inhibits thromboxane A2 synthetase in gel-filtered human platelets with an IC50 value of 4.5 +/- 0.5 nM (n = 4), whereas an inhibitory effect on cyclooxygenase is seen only at a much higher concentration (IC50: 240 microM). Radioligand-binding studies with [3H]SQ 29,548 in washed human platelets revealed that 36 blocks the prostaglandin H2/thromboxane A2 receptor with an IC50 of 19 +/- 5 nM (n = 5) and is therefore 85-fold more potent than another combined thromboxane A2 synthetase inhibitor/receptor antagonist, Ridogrel (4). Compound 36 inhibits the collagen-induced platelet aggregation in human platelet-rich plasma and whole blood with an EC50 of 1 microM (Ridogrel: 16 microM) and 100 nM, respectively, and was selected for further development.

Dipyridamole alone or combined with low-dose acetylsalicylic acid inhibits platelet aggregation in human whole blood ex vivo.

1. In a randomized, double-blind trial we compared the inhibition of the platelet-vessel wall interactions in whole blood ex vivo. There were four groups of 24 healthy volunteers each of whom were treated orally for 3.5 days with either 200 mg dipyridamole (sustained release preparation), 25 mg acetylsalicylic acid, both drugs combined or placebo twice daily. 2. The mean area of all platelets/aggregates was reduced by 6.2% +/- 4.2% (+/- s.e. mean) by placebo (n = 23), 19.8% +/- 6.7% by dipyridamole (n = 22), 53.7% +/- 4.9% by acetylsalicylic acid (n = 23) and 71.4% +/- 3.7% by the combination of both drugs (n = 24), when compared with total inhibition of aggregation by EGTA. Thus, low-dose acetylsalicylic acid inhibited aggregation (P less than 0.001). 3. Dipyridamole reduced the size of platelet aggregates (P less than 0.01, two-fold analysis of variance). The reduction was correlated with the individual dipyridamole plasma levels (P less than 0.05, analysis of covariance). The subgroup of large and very large thrombi being formed was also reduced by dipyridamole (P less than 0.05). 4. This ex vivo study demonstrates that dipyridamole alone inhibits formation of thrombi on subendothelial matrix and enhances the inhibitory effect of low dose acetylsalicylic acid in this model of thrombosis.

Effects of heparin on prostacyclin binding and platelet adenylate cyclase activity stimulated by iloprost and forskolin.

Heparin is known to impair the antiaggregatory effectiveness of prostacyclin (PGI2) for adenosine diphosphate-induced aggregation in citrated platelet rich plasma. Thus porcine mucosal heparin (PMH) from different commercial sources was investigated for its ability to compete with specific platelet prostacyclin binding. In concentrations up to 250 IU/ml PMH itself did not interfere with the binding of 3 X 10(-9) M 9-3H-PGI2-sodium salt to intact washed platelets. The displacement of specifically bound PGI2 observed by PMH (Ki 60 IU/ml) containing 4-chloro-m-cresol (4-CC) was caused by the preservative (Ki 4-CC 3.0 X 10(-4) M). Although 60 IU/ml PMH (free of 4-CC) have been shown to inhibit basal 3 X 10(-5) M forskolin-, and 10(-8)M Iloprost-stimulated adenylate cyclase in platelet membranes (-63%, -62%, -83% respectively), no effect of PMH on 3H-cyclic-AMP formation has been observed when intact platelets were studied by 3H-adenine prelabeling technique. There is also no evidence that the preincubation of PGI2 (5 X 10(-7) M) with 1000 IU/ml PMH might neutralize the effectiveness of this eicosanoid. In contrast, PGI2 preincubated with PMH (10 min, 37 degrees C) caused a more pronounced increase of platelet 3H-cyclic AMP (+65%) compared with PGI2 incubated in 5 X 10(-2) M Tris-HCl, pH 7.4. Thus our data provide no evidence that PMH interferes with i) specific platelet PGI2 binding, ii) PGI2-stimulated cyclic AMP synthesis or iii) neutralizes PGI2 by formation of a biologically less active PGI2-PMH complex.

The binding of 6-oxo-prostaglandin E1 to platelet prostanoid receptors.

6-oxo-PGE1 has been shown to cause a dose dependent stimulation of platelet adenylate cyclase (Km 1.6 X 10(-7)M). Since this compound also displaced specifically bound 9-3H-PGI2 from intact washed platelets (Ki 1.6 X 10(-6)M), the coupling to platelet PGI2 receptors has been suggested to account for the observed platelet cyclase stimulation. However, the Ki/Km-ratio of 6-oxo-PGE1 appears to be high (10.3) as compared to PGI2, ZK 36 374, and PGE1 (1.9, 2.4 and 3.1, resp.). Thus, one might expect that 6-oxo-PGE1 interacts with a heterogeneous population of platelet receptors mediating the same biological response, eg cyclase activation. In this study, we present evidence supporting this concept and report that 6-oxo-PGE1 also displaces specifically bound 3H-PGD2 from its platelet receptor (Ki 1.6 X 10(-5)M).

Platelet prostacyclin binding in coronary artery disease.

Reduced responsiveness of platelets to prostacyclin, reported in vitro in patients with coronary artery disease, has been thought to be a factor predisposing toward coronary thrombosis and vasospasm as a result of enhanced in vivo release of cyclic endoperoxides and thromboxane A2 by the platelets. In this study, specific binding of prostacyclin to intact platelets was determined in patients with coronary artery disease by direct binding studies using 9-3H-prostacyclin sodium salt. In addition, the inhibitory effect of prostacyclin on primary aggregation induced by adenosine diphosphate and cyclic adenosine monophosphate (cyclic AMP) accumulation stimulated by prostacyclin was examined. Twenty patients with angiographically documented coronary artery disease and stable angina, 8 patients with acute myocardial infarction, 14 healthy volunteers and 10 patients with normal angiograms were studied. In patients with stable angina, binding capacity and affinity of platelet prostacyclin binding sites and prostacyclin-induced cyclic AMP accumulation were not different from those of control subjects. In patients with acute myocardial infarction, however, binding capacity of platelet prostacyclin receptors was significantly reduced (0.69 +/- 0.45 versus 1.35 +/- 0.37 pmol/10(9) platelets, p = 0.001) and the postreceptor response, represented by platelet responsiveness to prostacyclin and prostacyclin-induced cyclic AMP synthesis, was impaired. Because all patients with myocardial infarction were receiving intravenous heparin and nitroglycerin, which might interfere with platelet prostacyclin binding, competition experiments were performed in vitro. Neither heparin (3 to 250 IU/ml) nor nitroglycerin (0.8 to 22 microM) displaced specifically bound 9-3H-prostacyclin. L-Epinephrine in concentrations up to 10 microM also exhibited no competition with specific platelet prostacyclin binding.(ABSTRACT TRUNCATED AT 250 WORDS)

Platelet prostacyclin binding in smokers.

Binding capacity (Bmax) and dissociation constant (KD) of platelet prostacyclin (PGI2) receptors in 23 male smokers and 14 nonsmokers have been determined by direct binding studies to investigate whether platelet prostacyclin binding is altered in smokers. In addition, the inhibitory effect of PGI2 (IC50) on adenosine diphosphate (ADP)-induced platelet aggregation was measured. As confirmed by discriminant analysis, 69% of the smokers had significantly different Bmax and KD. Binding capacity was increased in 12 smokers (Bmax + 74%), with a concomitant decrease in affinity (KD + 94%). In these volunteers, the postreceptor responses (PGI2-induced cyclic adenosine monophosphate [AMP] accumulation in platelet-rich plasma as well as IC50) did not differ from those of controls. In contrast, four smokers with considerably reduced PGI2 binding capacity (Bmax - 65%) exhibited a lower antiaggregatory effect (IC50 + 74%), although the affinity was slightly increased (KD - 61%). Nicotine, L-epinephrine, and prostaglandin E2 did not significantly compete with the binding of 9-3H-PGI2 sodium salt. The antiaggregatory effect of PGI2 on ADP-induced platelet aggregation, however, was inhibited by L-epinephrine (Ki 26 nmol/L) and prostaglandin E2 (Ki 230 nmol/L). Our data suggest that with respect to platelet PGI2 binding and in vitro responsiveness to PGI2, smokers are a heterogeneous population. Although increased binding capacity was observed in 50% of the smokers investigated, our data provide no evidence that a biologically relevant upregulation, for example with concomitant enhanced postreceptor reaction, occurs in smokers.

Identification of platelet inhibitor present in the melon (Cucurbitacea cucumis melo).

An active fraction was isolated from an aqueous melon extract (Cucurbitacea cucumis melo) and was shown that it inhibits human platelet aggregation induced by epinephrine, ADP, collagen, thrombin, sodium arachidonate, prostaglandin endoperoxide analogue U-46619 and PAF-acether. Identification of the active substance as adenosine was indicated by TLC which gave identical Rf value compared to adenosine, by the UV spectrum, because the inhibitory effect on platelet aggregation disappeared after the addition of adenosine-deaminase and because the substance under study and adenosine produced the same spectra in the mass spectroscopy.

Pharmacokinetics of oral dipyridamole (Persantine) and its effect on platelet adenosine uptake in man.

Two preparations of dipyridamole have been studied by oral administration to 11 normal volunteers. The plasma levels of dipyridamole and its glucuronide were determined simultaneously by high performance liquid chromatography. The instant form (I.F., 100 mg) was administered four times daily and the slow release preparation (SRP, 200 mg) twice daily, for 3 days. Multiple blood samples were collected on Days 1-4 to provide plasma for assay, and simultaneously, platelet rich plasma was prepared for ex vivo study of the effect of dipyridamole on platelet uptake of adenosine. The pharmacokinetics of absorption and distribution of dipyridamole were described using a two compartment model with lag time and prolonged absorption. Strong inhibition of the platelet adenosine uptake was observed at therapeutic plasma levels. The inhibition of platelet adenosine uptake may be related to some of the pharmacological properties of dipyridamole.