Jeffrey M. Bozarth

Tetrahydroquinoline derivatives as potent and selective factor XIa inhibitors.

Antithrombotic agents that are inhibitors of factor XIa (FXIa) have the potential to demonstrate robust efficacy with a low bleeding risk profile. Herein, we describe a series of tetrahydroquinoline (THQ) derivatives as FXIa inhibitors. Compound 1 was identified as a potent and selective tool compound for proof of concept studies. It exhibited excellent antithrombotic efficacy in rabbit thrombosis models and did not prolong bleeding times. This demonstrates proof of concept for the FXIa mechanism in animal models with a reversible, small molecule inhibitor.

Novel nonpeptide antiplatelet glycoprotein llb/llla receptor antagonist, DMP754: receptor binding affinity and specificity

ObjectiveTo define the antiplatelet efficacy and specificity of the glycoprotein llb/llla complex (GPIIb/llla) antagonist prodrug DMP754 and its free acid form, XV459. Methods and materialsDMP754 has an IC50 > 1 μmol/l, and, upon its conversion with esterases to its free acid form, demonstrated high potency (IC50 20–45 nmol/l) in inhibiting human platelet aggregation induced by 10 μmol/l adenosine diphosphate, 20 μg/ml collagen, 1 mmol/l epinephrine, 10 μmol/l platelet activating factor or 0.5 lU/ml thrombin. The in-vitro rate of hydrolysis of DMP754 or XV459 is much faster with human or canine liver esterases (t1/2 = 2.4–23 min) than with plasma esterases (t1,2 = 5.5–7.6 h). Platelet Gpllb/llla integrin binding affinity and specificity for XV459 were determined using cell binding/adhesion assays. ResultsThe range of IC50 values of XV459 in inhibiting platelet aggregation in platelet-rich plasma obtained from 12 subjects was 0.035–0.069 μmol/l with a mean IC50 of 0.050 ± 0.003 μmol/l. Additionally, XV459 inhibited platelets obtained from mongrel dogs, baboons, sheep, guinea pigs, and mice with IC50 in the range 0.024–0.06 μmol/l, and IC50 in the range 0.16–5.8 μmol/l in pigs, rabbits, and rats. XV459 inhibited [125l]-fibrinogen binding to activated human platelets with an IC50 of 0.011 ± 0.003 μmol/l. XV459 demonstrated a high degree of selectivity in specifically inhibiting fibrinogen binding to the platelet integrin, GPIIb/llla (IC50 = 0.00025 ± 0.00005 μmol/l) compared with inhibiting other integrins (αvβ3, IC50 > 10 μmol/l; or αvβ5, α5β1, or α5β1, for which the IC50 exceeded 100 μmol/l). ConclusionDMP754 is a potent antiplatelet agent in inhibiting platelet aggregation, and has a high specificity and affinity for human platelet GPIIb/llla receptors.

Phenylimidazoles as Potent and Selective Inhibitors of Coagulation Factor XIa with in Vivo Antithrombotic Activity

Novel inhibitors of FXIa containing an (S)-2-phenyl-1-(4-phenyl-1H-imidazol-2-yl)ethanamine core have been optimized to provide compound 16b, a potent, reversible inhibitor of FXIa (Ki = 0.3 nM) having in vivo antithrombotic efficacy in the rabbit AV-shunt thrombosis model (ID50 = 0.6 mg/kg + 1 mg kg(-1) h(-1)). Initial analog selection was informed by molecular modeling using compounds 11a and 11h overlaid onto the X-ray crystal structure of tetrahydroquinoline 3 complexed to FXIa. Further optimization was achieved by specific modifications derived from careful analysis of the X-ray crystal structure of the FXIa/11h complex. Compound 16b was well tolerated and enabled extensive pharmacologic evaluation of the FXIa mechanism up to the ID90 for thrombus inhibition.

Discovery of Novel P1 Groups for Coagulation Factor VIIa Inhibition Using Fragment-Based Screening

A multidisciplinary, fragment-based screening approach involving protein ensemble docking and biochemical and NMR assays is described. This approach led to the discovery of several structurally diverse, neutral surrogates for cationic factor VIIa P1 groups, which are generally associated with poor pharmacokinetic (PK) properties. Among the novel factor VIIa inhibitory fragments identified were aryl halides, lactams, and heterocycles. Crystallographic structures for several bound fragments were obtained, leading to the successful design of a potent factor VIIa inhibitor with a neutral lactam P1 and improved permeability.

XV454, a novel nonpeptide small-molecule platelet GIIb/IIIa antagonist with comparable platelet αIIbβ3-Binding kinetics to c7E3

XV454 demonstrated high potency (IC50 = 14-25 nM) in inhibiting human platelet aggregation induced by adenosine diphosphate (ADP, 10 microM), thrombin receptor agonist peptide (TRAP) (10 microM), or collagen (20 microg/ml). XV454 exhibited a high degree of selectivity for platelet alpha(IIb)beta3 in comparison with c7E3, which is a nonspecific antagonist for both alpha(IIb)beta3 and alpha(v)beta3. Both XV454 and c7E3 bind with high affinity to either activated (A) or unactivated (U) human, baboon, or canine platelets. XV454 binds with a relatively higher affinity [Kd = 0.5 nM (A), 0.6 nM (U)] as compared with c7E3 [Kd = 9.1 nM (A), 9.2 (U) nM]. XV454 demonstrated a tight association with human, baboon, and, to a lesser extent, with canine platelets (t(1/2) of dissociation = 110 +/- 6, 80 +/- 10, and 23 +/- 2 min, respectively). Both c7E3 and XV454 associate tightly with a slower dissociation rate with unactivated human platelets: t(1/2) of 42 and 116 min, respectively. In non-human primates, oral (0.1 mg/kg, p.o.) and intravenous (0.05 mg/kg, i.v. bolus administration of XV454 methyl ester pro-drug resulted a long-lasting maximal antiplatelet efficacy for < or = 72 h with significant but reversible prolongation of bleeding time and without effects on platelet count, clinical chemistry, or hemodynamic profile. In conclusion, XV454 represents a potent antiplatelet agent in inhibiting platelet aggregation along with a high affinity and relatively slow dissociation rate from human platelet GPIIb/IIIa receptors that allow a long-lasting antiplatelet efficacy after single i.v. or oral administration.

Oral antiplatelet efficacy and specificity of a novel nonpeptide platelet GPIIb/IIIa receptor antagonist, DMP 802

This study was undertaken to define the platelet glycoprotein alphaIIb beta3 integrin (GPII/IIIa) affinity, specificity, and oral antiplatelet efficacy of DMP 802, a small-molecule nonpeptide antiplatelet agent. Platelet GPIIb/IIIa integrin binding affinity and specificity for DMP 802 were determined by using binding and adhesion assays with cells from various species, including human. DMP 802 demonstrated a potent antiplatelet efficacy [median inhibitory concentration (IC50), 0.029 +/- 0.0042 microM] in inhibiting human platelet aggregation induced by 10 microM adenosine diphosphate (ADP), as assessed by light-transmittance aggregometry. DMP 802 inhibited 125I-fibrinogen binding to activated (ADP, epinephrine, and arachidonic acid at 100 microM each) gel purified human platelets with an IC50 of 0.012 +/- 0.003 microM. DMP 802 demonstrated tight association with unactivated human, baboon, or canine platelets (t(1/2) of dissociation, 32 +/- 2, 32 +/- 13, and 11 +/- 1 min, respectively). DMP 802 binds with high affinity to both unactivated and activated human platelets (Kd = 0.61 +/- 0.17, 0.57 +/- 0.21 nM, respectively). DMP 802 demonstrated species specificity in inhibiting platelet aggregation with IC50 values ranging from 0.025 to 0.092 microM (human, guinea pig, dog, swine, hamster) and 0.88-1.0 microM (rabbit and rat) in platelets obtained from these various species. DMP 802 demonstrated a high degree of specificity for platelet GPIIb/IIIa (alphaIIb/beta3) as compared with other integrins including alpha(v)beta3 (IC50, >10 microM), alpha(v)beta5 (IC50, >100 microM), alpha4beta1 (IC50, >100 microM), and alpha5beta1 (IC50, >10 microM). Oral antiplatelet efficacy of DMP 802 was examined after single oral (0.05-0.20 mg/kg) and after repeated oral dosing at 0.05 mg/kg daily for 5 days in mongrel dogs. Dose-dependent antiplatelet efficacy with an extended duration of antiplatelet efficacy was demonstrated based on ex vivo inhibition of platelet aggregation induced by 100 microM ADP. DMP 802 has an oral bioavailability of 14.9% in dogs. In conclusion, the alpha sulfonamide isoxazoline analog, DMP 802, is a novel oral antiplatelet agent with high affinity, relatively slow dissociation rate and specificity for human platelet GPIIb/IIIa receptors.

Using thrombelastography to determine the efficacy of the platelet glycoprotein IIb/IIIa antagonist, roxifiban, on platelet/fibrin-mediated clot dynamics in humans

The effect of platelet glycoprotein IIb/IIIa antagonists on the dynamics of platelet/fibrin clot formation and strength was determined using thrombelastography (TEG) under conditions of recalcification or tissue factor addition. In the present investigation, the effect of roxifiban (class I) on ex vivo clot dynamics using recalcified blood was tested in normal, healthy volunteers (n = 7) dosed with 1 mg BID roxifiban for 9 days. Roxifiban inhibited platelet aggregation induced by 20 μmol/l adenosine diphosphate by 60–90% but did not significantly affect any of the TEG parameters either at peak, trough, or subtrough drug levels. Addition of 30 nmol/l roxifiban free acid (XV459; which is ineffective by itself to modify TEG parameters) to human blood obtained from roxifiban-treated subjects resulted in 45–60% (P < 0.01) inhibition of clot strength (maximum amplitude), 90–100% (P < 0.01) inhibition of initial kinetic of clot development (angle α), and 50–70% (P < 0.01) inhibition of early clot initiation (K). These data suggest that a subthreshold blood level of 40–50 nmol/l roxifiban active form was achieved in those subjects, as estimated from an in vitro calibration with XV459. These data indicate (not studied) that roxifiban, at a targeted clinical dosing regimen, failed to achieve sufficient exposure to modulate platelet-mediated clot retraction.

Effect of thrombin inhibitors on platelet functions : comparative analysis of DuP 714 and hirudin

Since thrombin plays an important role in platelet-mediated arterial thrombosis, we have examined the antiplatelet activity of a synthetic thrombin inhibitor, DuP 714 (Ac-(D)Phe-Pro-boroArg), in comparison with that of the naturally occurring inhibitor hirudin. Hirudin was slightly more potent than DuP 714 in inhibiting thrombin-induced aggregation in washed human platelets (IC50s of 72 nM and 150 nM, respectively) and in inhibiting the secretion of plasminogen activator inhibitor-I from human platelets (IC50s of 300 nM and 900 nM, respectively). In contrast, DuP 714 was more potent than hirudin in inhibiting thrombin-induced [125I]fibrinogen binding to gel purified platelets, and in inhibiting thrombin-induced intracellular calcium mobilization in washed platelets. These results indicate that the tripeptide DuP 714 has comparable antiplatelet activity to the 65 amino acid hirudin. We conclude that DuP 714 may have clinical utility in the prevention of platelet-dependent, arterial thrombotic processes.

Inhibition of the thrombin-platelet reactions by DuP 714

The efficacy and specificity of a novel synthetic thrombin inhibitor, DuP 714, on thrombin-induced elevation of cytoplasmic calcium, fibrinogen binding and aggregation in human platelets were examined. Thrombin (0.5 U/ml) stimulated an increase in [125I]fibrinogen binding in gel-filtered platelets which was blocked by DuP 714 with an IC50 value of 2 nM. Thrombin (1 U/ml)-induced elevation of intracellular [Ca2+]i was also blocked by DuP 714 with an IC50 value of 67 nM. A much higher concentration of thrombin (25 U/ml) was used to stimulate aggregation with heparinized platelet-rich plasma. Under these conditions, micromolar concentrations of DuP 714 were needed to inhibit thrombin. In all of these preparations, DuP 714 at concentrations as high as 10(-5) M had no intrinsic effects and did not affect the responses induced by arachidonate, ADP, collagen, epinephrine, vasopressin and serotonin. These data indicate that DuP 714 is a potent and specific thrombin inhibitor capable of arresting the actions of thrombin on human fibrin formation and platelet aggregation and secretion. It may serve as a potential antithrombotic agent for various forms of thrombotic disorders.

Structure-Based Design of Macrocyclic Factor XIa Inhibitors: Discovery of the Macrocyclic Amide Linker

A novel series of macrocyclic FXIa inhibitors was designed based on our lead acyclic phenyl imidazole chemotype. Our initial macrocycles, which were double-digit nanomolar FXIa inhibitors, were further optimized with assistance from utilization of structure-based drug design and ligand bound X-ray crystal structures. This effort resulted in the discovery of a macrocyclic amide linker which was found to form a key hydrogen bond with the carbonyl of Leu41 in the FXIa active site, resulting in potent FXIa inhibitors. The macrocyclic FXIa series, exemplified by compound 16, had a FXIa Ki = 0.16 nM with potent anticoagulant activity in an in vitro clotting assay (aPTT EC1.5x = 0.27 μM) and excellent selectivity against the relevant blood coagulation enzymes.