Julie A. Krueger

Activation of farnesoid X receptor prevents atherosclerotic lesion formation in LDLR−/− and apoE−/− mice

The role of farnesoid X receptor (FXR) in the development of atherosclerosis has been unclear. Here, LDL receptor (LDLR−/−) or apolipoprotein E (apoE−/−) female or male mice were fed a Western diet and treated with a potent synthetic FXR agonist, WAY-362450. Activation of FXR blocked diet-induced hypertriglyceridemia and elevations of non-HDL cholesterol and produced a near complete inhibition of aortic lesion formation. WAY-362450 also induced small heterodimer partner (SHP) expression and repressed cholesterol 7α-hydroxylase (CYP7A1) and sterol 12 α-hydroxylase (CYP8B1) expression. To determine if SHP was essential for these protective activities, LDLR−/−SHP−/− and apoE−/−SHP−/− mice were similarly treated with WAY-362450. Surprisingly, a notable sex difference was observed in these mice. In male LDLR−/−SHP−/− or apoE−/−SHP−/− mice, WAY-362450 still repressed CYP7A1 and CYP8B1 expression by 10-fold and still strongly reduced non-HDL cholesterol levels and aortic lesion area. In contrast, in the female LDLR−/−SHP−/− or apoE−/−SHP−/− mice, WAY-362450 only slightly repressed CYP7A1 and CYP8B1 expression and did not reduce non-HDL cholesterol or aortic lesion size. WAY-362450 inhibition of hypertriglyceridemia remained intact in LDLR−/− or apoE−/− mice lacking SHP of both sexes. These results suggest that activation of FXR protects against atherosclerosis in the mouse, and this protective effect correlates with repression of bile acid synthetic genes, with mechanistic differences between male and female mice.

A synthetic farnesoid X receptor (FXR) agonist promotes cholesterol lowering in models of dyslipidemia

The nuclear hormone receptor farnesoid X receptor (FXR) plays a critical role in the regulation of bile acid, triglyceride (TG), and cholesterol homeostasis. WAY-362450 (FXR-450/XL335) is a potent synthetic FXR agonist as characterized in luciferase reporter assays and in mediating FXR target gene regulation in primary human and immortalized mouse hepatocytes. In vivo, WAY-362450 dose dependently decreased serum TG levels after 7 days of oral dosing in western diet-fed low-density lipoprotein receptor-/- mice and in the diabetic mouse strains KK-Ay and db/db comparable to that achieved with the peroxisome proliferator activated receptor-alpha agonist, fenofibrate. WAY-362450 treatment also reduced serum cholesterol levels via reductions in LDLc, VLDLc, and HDLc lipoprotein fractions that were not accompanied by hepatic cholesterol accumulation. This cholesterol lowering was dependent on FXR as demonstrated in a hypothyroid-induced hypercholesterolemia setting in FXR-/- mice. In fructose-fed models, WAY-362450 also decreased TG and VLDLc levels in rats and hamsters but significantly increased HDLc levels in rats while reducing HDLc levels in hamsters. The differential effect of WAY-362450 on HDLc is likely due to a murine-specific induction of endothelial lipase and scavenger receptor-BI that does not occur in rats. These studies demonstrate a consistent ability of WAY-362450 to lower both serum TG and cholesterol levels and suggest that synthetic FXR agonists may have clinical utility in the treatment of mixed dyslipidemia.

L-374,087, an efficacious, orally bioavailable, pyridinone acetamide thrombin inhibitor

Replacement of the amidinopiperidine P1 group of 3-benzylsulfonylamino-6-methyl-2-pyridinone acetamide thrombin inhibitor L-373,890 (2) with a mildly basic 5-linked 2-amino-6-methylpyridine results in an equipotent compound L-374,087 (5, Ki = 0.5 nM). Compound 5 is highly selective for thrombin over trypsin, is efficacious in the rat ferric chloride model of arterial thrombosis and is orally bioavailable in dogs and cynomolgus monkeys. The structural basis for the critical importance of both methyl groups in 5 was confirmed by X-ray crystallography.

The Gap Junction Modifier, GAP-134 [(2S,4R)-1-(2-Aminoacetyl)-4-benzamido-pyrrolidine-2-carboxylic Acid], Improves Conduction and Reduces Atrial Fibrillation/Flutter in the Canine Sterile Pericarditis Model

Gap junction uncoupling can alter conduction pathways and promote cardiac re-entry mechanisms that potentiate many supraventricular arrhythmias, such as atrial fibrillation (AF) and atrial flutter (AFL). Our objective was to determine whether GAP-134 [(2S,4R)-1-(2-aminoacetyl)-4-benzamido-pyrrolidine-2-carboxylic acid], a small dipeptide gap junction modifier, can improve conduction and ultimately prevent AF/AFL. In rat atrial strips subjected to metabolic stress, GAP-134 prevented significantly conduction velocity slowing at 10 nM compared with vehicle (p < 0.01). In the canine sterile pericarditis model, conduction time (CT; n = 5), atrial effective refractory period (AERP; n = 3), and AF/AFL duration/inducibility (n = 16) were measured 2 to 3 days postoperatively in conscious dogs. CT was significantly faster after GAP-134 infusion (average plasma concentration, 250 nM) at cycle lengths of 300 ms (66.2 ± 1.0 versus 62.0 ± 1.0 ms; p < 0.001) and 200 ms (64.4 ± 0.9 versus 61.0 ± 1.3 ms; p < 0.001). No significant changes in AERP were noted after GAP-134 infusion. The mean number of AF/AFL inductions per animal was significantly decreased after GAP-134 infusion (2.7 ± 0.6 versus 1.6 ± 0.8; p < 0.01), with total AF/AFL burden being decreased from 12,280 to 6063 s. Western blot experiments showed no change in connexin 43 expression. At concentrations exceeding those described in the AF/AFL experiments, GAP-134 had no effect on heart rate, blood pressure, or any electrocardiogram parameters. In conclusion, GAP-134 shows consistent efficacy on measures of conduction and AF/AFL inducibility in the canine sterile pericarditis model. These findings, along with its oral bioavailability, underscore its potential antiarrhythmic efficacy.

Pharmacokinetic optimization of 3-amino-6-chloropyrazinone acetamide thrombin inhibitors. Implementation of P3 pyridine N-oxides to deliver an orally bioavailable series containing P1 N-benzylamides.

In this manuscript we demonstrate that a modification principally directed toward the improvement of the aqueous solubility (i.e., introduction a P3 pyridine N-oxide) of the previous lead compound afforded a new series of potent orally bioavailable P1 N-benzylamide thrombin inhibitors. An expedited investigation of the P1 SAR with respect to oral bioavailability, plasma half-life, and human liver microsome stability revealed 5 as the best candidate for advanced evaluation.

Unexpected enhancement of thrombin inhibitor potency with o-aminoalkylbenzylamides in the P1 position.

Thrombin inhibitors incorporating o-aminoalkylbenzylamides in the P1 position were designed, synthesized and found to have enhanced potency and selectivity in several different structural classes. X-ray crystallographic analysis of compound 24 bound in the alpha-thrombin-hirugen complex provides an explanation for these unanticipated results.

Neutralization of plasminogen activator inhibitor I (PAI-1) by the synthetic antagonist PAI-749 via a dual mechanism of action.

PAI-749 is a potent and selective synthetic antagonist of plasminogen activator inhibitor 1 (PAI-1) that preserved tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA) activities in the presence of PAI-1 (IC50 values, 157 and 87 nM, respectively). The fluorescence (Fl) of fluorophore-tagged PAI-1 (PAI-NBD119) was quenched by PAI-749; the apparent Kd (254 nM) was similar to the IC50 (140 nM) for PAI-NBD119 inactivation. PAI-749 analogs displayed the same potency rank order for neutralizing PAI-1 activity and perturbing PAI-NBD119 Fl; hence, binding of PAI-749 to PAI-1 and inactivation of PAI-1 activity are tightly linked. Exposure of PAI-1 to PAI-749 for 5 min (sufficient for full inactivation) followed by PAI-749 sequestration with Tween 80 micelles yielded active PAI-1; thus, PAI-749 did not irreversibly inactivate PAI-1, a known metastable protein. Treatment of PAI-1 with a PAI-749 homolog (producing less assay interference) blocked the ability of PAI-1 to displace p-aminobenzamidine from the uPA active site. Consistent with this observation, PAI-749 abolished formation of the SDS-stable tPA/PAI-1 complex. PAI-749-mediated neutralization of PAI-1 was associated with induction of PAI-1 polymerization as assessed by native gel electrophoresis. PAI-749 did not turn PAI-1 into a substrate for tPA; however, PAI-749 promoted plasmin-mediated degradation of PAI-1. In conclusion, PAI-1 inactivation by PAI-749 using purified components can result from a dual mechanism of action. First, PAI-749 binds directly to PAI-1, blocks PAI-1 from accessing the active site of tPA, and abrogates formation of the SDS-stable tPA/PAI-1 complex. Second, binding of PAI-749 to PAI-1 renders PAI-1 vulnerable to plasmin-mediated proteolytic degradation.

Azaindoles: Moderately basic P1 groups for enhancing the selectivity of thrombin inhibitors

Starting from a 2-amino-6-methylpyridine P1 group and following a strategy of enlarging it whilst reducing its polarity, we have developed a series of potent, moderately basic azaindoles which are intrinsically much more selective for thrombin versus trypsin. Certain pyrazinone acetamide azaindole derivatives have pharmacokinetic parameters after oral administration to dogs, or efficacy in vitro, comparable to an optimized pyrazinone acetamide 2-amino-6-methylpyridine derivative.

Design and synthesis of potent and selective macrocyclic thrombin inhibitors

A series of potent and selective proline- and pyrazinone-based macrocyclic thrombin inhibitors is described. Detailed SAR studies led to the incorporation of specific functional groups in the tether that enhanced functional activity against thrombin and provided exquisite selectivity against trypsin and tPA. X-ray crystallography and molecular modeling studies revealed the inhibitor-enzyme interactions responsible for this selectivity.

Bicyclic pyridones as potent, efficacious and orally bioavailable thrombin inhibitors.

A new class of conformationally constrained thrombin inhibitors is described. These compounds contain a unique bicyclic pyridone scaffold which serves as a P3P2 dipeptide surrogate. The synthesis and antithrombotic activity of these inhibitors is reported.