Laura A. Price

Biomarker Optimization to Track the Antithrombotic and Hemostatic Effects of Clopidogrel in Rats

We determined the dose response of the ADP antagonist clopidogrel (0.3-50 mg/kg p.o.) in rat models of thrombosis and provoked bleeding and correlated these activities to ex vivo platelet activation. Carotid artery thrombosis was induced by FeCl2. Bleeding time was measured by mesenteric vessel puncture and renal cortex or cuticle incision. Platelet biomarkers included standard ADP-induced aggregation, P2Y12 receptor occupancy, and phosphorylation of vasodilator-stimulated phosphoprotein. Clopidogrel decreased thrombus weight up to 78%, caused maximal prolongation of cuticle and mesenteric bleeding, but had little effect on renal bleeds. Due to the steep mesenteric dose response, further comparisons concentrated on cuticle bleeding. The half-maximal inhibitory dose (ED50) for thrombus reduction was 2.4 ± 0.4 mg/kg, with 10 mg/kg providing optimal blood flow preservation and thrombus reduction. The ED50 for bleeding was 10.5 ± 3.4 mg/kg. Increased bleeding was intermediate (3-fold) at 10 mg/kg and maximal (6-fold) at 30 mg/kg. All biomarkers were affected, but with differing sensitivity. ED50s for peak platelet aggregation to 10 μM ADP (11.9 ± 0.4 mg/kg) and the vasodilator-stimulated phosphoprotein index (16.4 ± 1.3 mg/kg) approximated the higher ED50 for bleeding. ED50s for ligand binding (3.0 ± 0.3 mg/kg) and late aggregation (5.1 ± 0.4 mg/kg) better matched the lower ED50 for antithrombotic activity. Aspirin exerted lesser effects on bleeding (42-70% increase in all models) and thrombosis (24% inhibition). In summary, antithrombotic doses of clopidogrel have limited effects on bleeding and standard measures of platelet aggregation. Other biomarkers may be more sensitive for tracking antithrombotic efficacy.

Engineering of a Novel Anti-CD40L Domain Antibody for Treatment of Autoimmune Diseases

CD40–CD40L interactions play a critical role in regulating immune responses. Blockade of CD40L by Abs, such as the anti-CD40L Ab 5c8, demonstrated positive clinical effects in patients with autoimmune diseases; however, incidents of thromboembolism (TE) precluded further development of these molecules. In this study, we examined the role of the Fc domain interaction with FcγRs in modulating platelet activation and potential for TE. Our results show that the interaction of the 5c8 wild-type IgG1 Fc domain with FcγRs is responsible for platelet activation, as measured by induction of PAC-1 and CD62P. A version of 5c8 with a mutated IgG1 tail was identified that showed minimal FcγR binding and platelet activation while maintaining full binding to CD40L. To address whether Fc effector function is required for immunosuppression, a potent Ab fragment, termed a “domain Ab” (dAb), against murine CD40L was identified and fused to a murine IgG1 Fc domain containing a D265A mutation that lacks Fc effector function. In vitro, this dAb–Fc demonstrated comparable potency to the benchmark mAb MR-1 in inhibiting B cell and dendritic cell activation. Furthermore, the anti-CD40L dAb–Fc exhibited a notable efficacy comparable to MR-1 in various preclinical models, such as keyhole limpet hemocyanin–induced Ab responses, alloantigen-induced T cell proliferation, “heart-to-ear” transplantation, and NZB × NZW F1 spontaneous lupus. Thus, our data show that immunosuppression and TE can be uncoupled and that a CD40L dAb with an inert Fc tail is expected to be efficacious for treating autoimmune diseases, with reduced risk for TE.

Discovery of 2-(phenoxypyridine)-3-phenylureas as small molecule P2Y1 antagonists.

Two distinct G protein-coupled purinergic receptors, P2Y1 and P2Y12, mediate ADP-driven platelet activation. The clinical effectiveness of P2Y12 blockade is well established. Recent preclinical data suggest that P2Y1 and P2Y12 inhibition provide equivalent antithrombotic efficacy, while targeting P2Y1 has the potential for reduced bleeding liability. In this account, the discovery of a 2-(phenoxypyridine)-3-phenylurea chemotype that inhibited ADP-mediated platelet aggregation in human blood samples is described. Optimization of this series led to the identification of compound 16, 1-(2-(2-tert-butylphenoxy)pyridin-3-yl)-3-4-(trifluoromethoxy)phenylurea, which demonstrated a 68 ± 7% thrombus weight reduction in an established rat arterial thrombosis model (10 mg/kg plus 10 mg/kg/h) while only prolonging cuticle and mesenteric bleeding times by 3.3- and 3.1-fold, respectively, in provoked rat bleeding time models. These results suggest that a P2Y1 antagonist could potentially provide a safe and efficacious antithrombotic profile.

Conformationally Constrained ortho-Anilino Diaryl Ureas: Discovery of 1-(2-(1′-Neopentylspiro[indoline-3,4′-piperidine]-1-yl)phenyl)-3-(4-(trifluoromethoxy)phenyl)urea, a Potent, Selective, and Bioavailable P2Y1 Antagonist

Preclinical antithrombotic efficacy and bleeding models have demonstrated that P2Y1 antagonists are efficacious as antiplatelet agents and may offer a safety advantage over P2Y12 antagonists in terms of reduced bleeding liabilities. In this article, we describe the structural modification of the tert-butyl phenoxy portion of lead compound 1 and the subsequent discovery of a novel series of conformationally constrained ortho-anilino diaryl ureas. In particular, spiropiperidine indoline-substituted diaryl ureas are described as potent, orally bioavailable small-molecule P2Y1 antagonists with improved activity in functional assays and improved oral bioavailability in rats. Homology modeling and rat PK/PD studies on benchmark compound 3l will also be presented. Compound 3l was our first P2Y1 antagonist to demonstrate a robust oral antithrombotic effect with mild bleeding liability in the rat thrombosis and hemostasis models.

New azole antagonists with high affinity for the P2Y(1) receptor.

Five-membered-ring heterocyclic urea mimics have been found to be potent and selective antagonists of the P2Y1 receptor. SAR of the various heterocyclic replacements is presented, as well as side-chain SAR of the more potent thiadiazole ring system which leads to thiadiazole 4c as a new antiplatelet agent.

Discovery of 4-Aryl-7-Hydroxyindoline-Based P2Y1 Antagonists as Novel Antiplatelet Agents

Adenosine diphosphate (ADP)-mediated platelet aggregation is signaled through two distinct G protein-coupled receptors (GPCR) on the platelet surface: P2Y12 and P2Y1. Blocking P2Y12 receptor is a clinically well-validated strategy for antithrombotic therapy. P2Y1 antagonists have been shown to have the potential to provide equivalent antithrombotic efficacy as P2Y12 inhibitors with reduced bleeding in preclinical animal models. We have previously reported the discovery of a potent and orally bioavailable P2Y1 antagonist, 1. This paper describes further optimization of 1 by introducing 4-aryl groups at the hydroxylindoline in two series. In the neutral series, 10q was identified with excellent potency and desirable pharmacokinetic (PK) profile. It also demonstrated similar antithrombotic efficacy with less bleeding compared with the known P2Y12 antagonist prasugrel in rabbit efficacy/bleeding models. In the basic series, 20c (BMS-884775) was discovered with an improved PK and liability profile over 1. These results support P2Y1 antagonism as a promising new antiplatelet target.

Identification of 1-{2-[4-chloro-1′-(2,2-dimethylpropyl)-7-hydroxy-1,2-dihydrospiro[indole-3,4′-piperidine]-1-yl]phenyl}-3-{5-chloro-[1,3]thiazolo[5,4-b]pyridin-2-yl}urea, a potent, efficacious and orally bioavailable P2Y1 antagonist as an antiplatelet agent

Spiropiperidine indoline-substituted diaryl ureas had been identified as antagonists of the P2Y1 receptor. Enhancements in potency were realized through the introduction of a 7-hydroxyl substitution on the spiropiperidinylindoline chemotype. SAR studies were conducted to improve PK and potency, resulting in the identification of compound 3e, a potent, orally bioavailable P2Y1 antagonist with a suitable PK profile in preclinical species. Compound 3e demonstrated a robust antithrombotic effect in vivo and improved bleeding risk profile compared to the P2Y12 antagonist clopidogrel in rat efficacy/bleeding models.

2-Aminothiazole based P2Y1 antagonists as novel antiplatelet agents

ADP receptors, P2Y1 and P2Y12 have been recognized as potential targets for antithrombotic drugs. A series of P2Y1 antagonists that contain 2-aminothiazoles as urea surrogates were discovered. Extensive SAR of the thiazole ring is described. The most potent compound 7j showed good P2Y1 binding (Ki=12nM), moderate antagonism of platelet aggregation (PA IC50=5.2μM) and acceptable PK in rats.

Discovery of diarylurea P2Y1 antagonists with improved aqueous solubility

Preclinical data suggests that P2Y1 antagonists, such as diarylurea compound 1, may provide antithrombotic efficacy similar to P2Y12 antagonists and may have the potential of providing reduced bleeding liabilities. This manuscript describes a series of diarylureas bearing solublizing amine side chains as potent P2Y1 antagonists. Among them, compounds 2l and 3h had improved aqueous solubility and maintained antiplatelet activity compared with compound 1. Compound 2l was moderately efficacious in both rat and rabbit thrombosis models and had a moderate prolongation of bleeding time in rats similar to that of compound 1.

Fc-Silent Anti-CD154 Domain Antibody Effectively Prevents Nonhuman Primate Renal Allograft Rejection

The advent of costimulation blockade provides the prospect for targeted therapy with improved graft survival in transplant patients. Perhaps the most effective costimulation blockade in experimental models is the use of reagents to block the CD40/CD154 pathway. Unfortunately, successful clinical translation of anti‐CD154 therapy has not been achieved. In an attempt to develop an agent that is as effective as previous CD154 blocking antibodies but lacks the risk of thromboembolism, we evaluated the efficacy and safety of a novel anti‐human CD154 domain antibody (dAb, BMS‐986004). The anti‐CD154 dAb effectively blocked CD40‐CD154 interactions but lacked crystallizable fragment (Fc) binding activity and resultant platelet activation. In a nonhuman primate kidney transplant model, anti‐CD154 dAb was safe and efficacious, significantly prolonging allograft survival without evidence of thromboembolism (Median survival time 103 days). The combination of anti‐CD154 dAb and conventional immunosuppression synergized to effectively control allograft rejection (Median survival time 397 days). Furthermore, anti‐CD154 dAb treatment increased the frequency of CD4+CD25+Foxp3+ regulatory T cells. This study demonstrates that the use of a novel anti‐CD154 dAb that lacks Fc binding activity is safe without evidence of thromboembolism and is equally as potent as previous anti‐CD154 agents at prolonging renal allograft survival in a nonhuman primate preclinical model.