Preclinical studies of RUC-4, a novel platelet αIIbβ3 antagonist, in non-human primates and with human platelets

Abstract

Introduction: We are developing the novel αIIbβ3 antagonist, RUC-4, for subcutaneously (SC)-administered first-point-of-medical-contact treatment for ST Segment Elevated Myocardial Infarction (STEMI). Methods: We studied the: 1. pharmacokinetics (PK) of RUC-4 at 1.0, 1.93, and 3.86 mg/kg IV, IM, and SC in non-human primates (NHPs); 2. impact of aspirin on RUC-4 IC50 in human platelet-rich plasma (PRP); 3. effect of different anticoagulants on the RUC-4 IC50 in human PRP; and 4. relationship between αIIbβ3 receptor blockade by RUC-4 and inhibition of ADP-induced platelet aggregation. Results: 1. All doses of RUC-4 were well tolerated, but animals demonstrated variable temporary bruising. IM and SC RUC-4 reached dose-dependent peak levels within 5–15 min, with T½ s between 0.28 and 0.56 hrs. Platelet aggregation studies in NHPs receiving IM RUC-4 demonstrated >80% inhibition of the initial slope of ADP-induced aggregation with all 3 doses 30 minutes post-dosing, with subsequent dose-dependent loss of inhibition over 4–5 hours. 2. The RUC-4 IC50 for ADP-induced platelet aggregation was unaffected by aspirin treatment (40±9 nM vs. 37±5 nM; p=0.39). 3. The RUC-4 IC50 was significantly higher in PRP prepared from PPACK-anticoagulated blood compared to citrate-anticoagulated blood using either TRAP (122±17 vs. 66±25 nM; p=0.05; n=4) or ADP (102±22 vs. 54±13; p<0.001; n=5). 4. There was a close correspondence between receptor blockade and inhibition of ADP-induced platelet aggregation, with aggregation inhibition beginning with ~40% receptor blockade and becoming nearly complete at >80% receptor blockade. Discussion: Based on these results and others, RUC-4 has now progressed to formal preclinical toxicology studies.

Application of Auxiliary VerifyNow Point-of-Care Assays to Assess the Pharmacodynamics of RUC-4, a Novel αIIbβ3 Receptor Antagonist

“…We previously reported that incubating whole blood with increasing concentrations of RUC-4 results in a decrease in the ability of PAC1 to bind to ADP-activated platelets, reflecting increased occupancy of αIIbβ3 receptors. 24 In the current study we assessed the effect of RUC-4 on PAC1 binding in parallel studies with both whole blood and PRP and correlated the results with the effects of RUC-4 on the VN PRUTest iso-TRAP and ADP þ PGE 1 assays and on ADP (20 µM)-induced LTA of PRP (►Fig. 2).…”

“…Nonetheless, the PrSl correlates well with results based on maximal aggregation and final aggregation in the in vitro studies (►Supplementary Figure S1) and maximal aggregations in the in vivo studies. 24…”

“…It has a unique mechanism of action that locks the receptor in its inactive conformation. [21][22][23][24] In a Phase 1 study conducted in healthy volunteers and stable coronary artery disease patients on aspirin, RUC-4 produced high grade inhibition of ADP (20 μM)-induced platelet aggregation as measured by light transmission aggregometry (LTA) within 15 minutes of SC administration, with rapid return of platelet function over the next 1-2 hours. 25 LTA is considered the established gold standard assay to assess the potency of antiplatelet agents because of its association with clinical outcomes.…”

“…26 We previously showed that citrate anticoagulation also enhances RUC-4's measured antiplatelet effects. 24 As a result, in the Phase 1 study of RUC-4, LTA was performed with blood anticoagulated with PPACK. 25 In the present study, we endeavored to study the effect of RUC-4 and the P2Y12 antagonist ticagrelor in vitro using the ADP þ PGE 1 , iso-TRAP, and base channel assays in the PRUtest and P2Y12 cartridges.…”

Insights into Protein–Ligand Interactions in Integrin Complexes: Advances in Structure Determinations

“…In fact, introducing a ring N (pyridine) yielded 15 (RUC-4), a potent antagonist (e.g., IC 50 for ADP-induced aggregation of untreated platelets is 40 ± 9 nM) that showed >60 mg/mL solubility in water with properties suitable for autoinjection . A preclinical study with this compound has been reported, and a Phase I clinical trial as potential treatment for myocardial infarction has been initiated …”